Does cooling affect the lipid composition of parasite free-living stages?

Does cooling affect the lipid composition of parasite free-living stages? | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 14 February 2025 V1 Latest version Share on Does cooling affect the lipid composition of parasite free-living stages? Authors : Kseniia Savina 0000-0002-9246-5475 [email protected] , Misha Gopko 0000-0002-1525-6557 , Viktor Voronin , Svetlana Murzina , and Ekaterina Mironova 0000-0002-9557-6031 Authors Info & Affiliations https://doi.org/10.22541/au.173952585.59301606/v1 176 views 110 downloads Contents Abstract Abstract Introduction Materials and methods Results Discussion References Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Temperature significantly influences the physiology of poikilotherms. However, the effect of short-term cooling on their lipids remains unknown. We investigated how cooling (~4 hours) affects the lipidome of Diplostomum pseudospathaceum cercariae, a free-living stage of a common freshwater trematode. Samples of cercariae were either cooled (up to 0 °C) or uncooled during collection and then analyzed using high-performance thin-layer chromatography and gas chromatography. Cooling did not significantly affect the composition and quantity of lipid classes and fatty acids. Contrary to our hypotheses, cooling did not increase the levels of total lipids and polyunsaturated fatty acids. This suggests that cercariae lack the lipidomic adjustments to low temperatures for maintaining optimal membrane fluidity that characterize many other organisms. Lipid analysis revealed essential fatty acids (13% of sum), highlighting the potential nutritional importance of cercariae. Unlike other cercariae species, D. pseudospathaceum showed high levels of triacylglycerols and waxes (10% and 18% of total lipids, respectively). These findings enhance understanding of cercarial lipid metabolism and support using cooling for lipid sampling. In addition, we provide important data on the dry weight of cercariae and methodological recommendations for their sampling, aiming to facilitate future research on these understudied organisms. Does cooling affect the lipid composition of parasite free-living stages? Abstract Temperature significantly influences the physiology of poikilotherms. However, the effect of short-term cooling on their lipids remains unknown. We investigated how cooling (~4 hours) affects the lipidome of Diplostomum pseudospathaceum cercariae, a free-living stage of a common freshwater trematode. Samples of cercariae were either cooled (up to 0 °C) or uncooled during collection and then analyzed using high-performance thin-layer chromatography and gas chromatography. Cooling did not significantly affect the composition and quantity of lipid classes and fatty acids. Contrary to our hypotheses, cooling did not increase the levels of total lipids and polyunsaturated fatty acids. This suggests that cercariae lack the lipidomic adjustments to low temperatures for maintaining optimal membrane fluidity that characterize many other organisms. Lipid analysis revealed essential fatty acids (13% of sum), highlighting the potential nutritional importance of cercariae. Unlike other cercariae species, D. pseudospathaceum showed high levels of triacylglycerols and waxes (10% and 18% of total lipids, respectively). These findings enhance understanding of cercarial lipid metabolism and support using cooling for lipid sampling. In addition, we provide important data on the dry weight of cercariae and methodological recommendations for their sampling, aiming to facilitate future research on these understudied organisms. Keywords : cercariae, trematode, lipidome, essential fatty acids, dry weight. Introduction Temperature is a key environmental factor and even moderate changes in it can alter physiology and biochemical composition in many organisms (Lee et al., 2003; Lee et al., 2017; Guschina & Harwood, 2006; Morley, 2011). The adaptations of poikilotherms to temperature shifts are primarily related to their lipid composition (Wu et al., 2023). A common adaptation to a decrease in temperature, which allows the maintenance of optimal membrane fluidity, is an increase in fatty acid unsaturation (Hazel & Williams, 1990; Hazel, 1995; Guschina & Harwood, 2006). Some poikilotherms can do it in a relatively short period, such as one hour (Jones et al., 1993) as an emergency adjustment to a sharp drop in temperature (Murata, 1989). Previous research on lipids has focused on long-term cooling in aquatic invertebrates (Nanton & Castell, 1999; Schlechtriem et al., 2006; Pond et al., 2014; Lee et al., 2017; Lee et al., 2020) or freezing (Morris, 1972; Ohman, 1996; Schariter et al. 2002). Nevertheless, the influence of short-term cooling, which organisms may experience in nature, on the lipidome remains unexplored. The metabolism of free-living stages of parasites, e.g., trematode cercariae, changes with temperature, which can influence their survival and parasite transmission. The available data concern glycogen utilization (Morley, 2011), while information about temperature effects on lipids in cercariae is scarce (Schariter et al., 2002). It is also unknown whether cercariae have similar adaptations of lipid composition to cooling as free-living organisms. In general, the composition and function of lipids in parasites remain less clear than in free-living organisms. It has been suggested that trematode cercariae mainly depend on glycogen storage as an energy source for movement and survival (Lawson & Wilson, 1980; Pechenik & Fried, 1995) rather than on lipids (Frayha & Smyth, 1983). Reserves of lipids, particularly triacylglycerols, in cercariae were supposed to diminish with time (Fokina et al., 2018; see, however, Fried et al., 1998). Moreover, Bexkens et al. (2019) could not detect any genes encoding enzymes required for energy production from lipids in Schistosoma mansoni . Therefore, the role of lipids as an energy source in cercariae is debatable. Lipids may be important for parasite development, survival and infectivity (Furlong & Caulfield 1988; Marsit et al., 2000; Schariter et al., 2002; Fokina et al., 2018). However, data on lipid composition and functions in cercariae are fragmentary and ambiguous. They mainly concern neutral lipids (Smith et al., 1966; Furlong & Caulfield 1988; Muller et al., 1999; Marsit et al., 2000, 2000a; Schariter et al., 2002; Fokina et al., 2018), while information on fatty acids profiles is scarce (Giera et al., 2018; Fokina et al., 2018; McKee et al., 2020). Besides parasite fitness, the lipid composition of cercariae can affect aquatic food webs. Cercariae are produced in great numbers by the first intermediate hosts (molluscs) and can be consumed by various predators and detritivores (Johnson et al., 2010; Mironova et al., 2019; McKee et al., 2020). Cercariae are likely rich in nutrients (e.g., polyunsaturated fatty acids) essential for the growth and reproduction of their predators (Mironova et al., 2019, 2020; McKee et al., 2020). Since the role of parasites in aquatic food webs is potentially enormous (Lafferty et al., 2008), studies on the cercariae lipidome are needed to quantify nutrient flows and energy transfer. Different approaches are used to collect cercariae for lipid analysis: centrifugation, passing water with cercariae through a mesh or a filter, and sedimentation. The latter is typically performed at 4 °C (Marsit et al., 2000, 2000a; Schariter et al., 2002) or on ice (Maloney et al., 1990; Giera et al., 2018) to accelerate the settling of cercariae. It is unclear whether cooling alters the lipid composition of cercariae because the available studies focus on the effect of freezing (Schariter et al., 2002). The lipid analysis of cercariae is problematic because it typically requires high-density samples (thousands of cercariae in a few milliliters), making sample collection very laborious. Additionally, the interpretation and comparison of lipid analysis results are often hampered by the lack of important information, such as the number and environmental background of hosts used as a source of cercariae, temperature, acclimation conditions, and the number of replicates. In this study, we propose methodological recommendations to facilitate cercariae sampling for lipid analysis and the interpretation of lipid analysis results. We chose Diplostomum pseudospathaceum , a common trematode in freshwater ecosystems (Shigin, 1986; Valtonen & Gibson, 1997), infecting snails (first intermediate host), fishes (second intermediate host), and fish-eating birds (definitive host). We studied the lipidome of this species for the first time and examined how short-term cooling (~4 h) affects the lipid composition of cercariae. Our hypotheses were as follows: (i) Cooled cercariae contain more total lipids than uncooled ones due to slower lipid utilization. (ii) In cooled cercariae, the absolute and relative amounts of polyunsaturated fatty acids (PUFA) are higher than in uncooled ones. Materials and methods Collection of cercariae We collected pond snails ( Lymnaea stagnalis ) in November 2023 from the lake Divnoe east of Moscow (55.762, 38.089). In the laboratory, we checked which snails shed cercariae of trematode D. pseudospathaceum . Cercariae were identified morphologically using the key by Faltýnková et al. (2007). To prevent active cercariae shedding, the infected snails were kept in dechlorinated water at 12 °C for about four months until the start of cercariae collection. During the maintenance and collection of cercariae, snails were fed by Lactuca sativa . To stimulate cercariae emergence, the infected snails were individually placed in 300-ml glasses filled with water and left at room temperature for about 24 hours. Then, mucus, faeces and lettuce were removed by a pipette from the water with cercariae and filtered through a 250-µm mesh. Cercariae of D. pseudospathaceum freely passed through such mesh. We used cercariae from 1–4 snails. These 600–900 ml samples were filtered again through the 50-µm mesh to immobilize cercariae via their division into tails and bodies. Then, some samples were processed with cooling, while others at room temperature. Cooled samples in jars were kept in a freezer (−20 °C) for 2 h and in a refrigerator (4 °C) for 2 h to prevent the freezing of the sample (in three samples cooling protocol differed, Supporting information). Overall, the temperature of cooled samples was up to 0 °C. Cooling ensured that most of cercariae settled at the bottom. Then, the upper layers of the water were removed and sedimented cercariae were further processed. The uncooled samples were left at room temperature (20 °C) in the darkness for 3 h to ensure that cercariae in both treatments had a similar age. Afterwards, all cercariae samples were centrifuged at 13000 RPM for 5 min in 15-ml tubes at 4 °C and 20 °C in the cooled and uncooled treatment, respectively. After each centrifugation cycle, the upper layer of water was decanted to decrease the total volume of the sample. Centrifugation took around 1.6 h until the sample volume reached several milliliters. This cercariae concentrate was put in several 1.5-ml tubes and centrifuged at 8600 RPM for 1 min at 20 °C several times. To count the number of cercariae in each sample, the clot of cercariae was carefully mixed and 10–30 µl of suspension was sampled. Finally, 1.5–2 ml of suspension was placed in a 5-ml Eppendorf plastic tube or a glass vial with 3 ml of a chloroform-methanol (2:1) extraction mixture and frozen at −20 °C till lipid analysis (figure 1). In total, we collected 18 samples of D. pseudospathaceum cercariae (9 cooled, 9 uncooled) (Supporting information). From six to ten replicates were counted under the microscope. Since cercariae broke into furcal tails and bodies, these parts were counted separately. The number of furcae and bodies in the sample often differed, therefore, the ‘extra’ parts were counted as half a cercaria when calculating the total number of cercariae in the sample. To roughly estimate the amount of lipids that could be lost during centrifugation, we collected 4 control samples. The supernatant from the tube at the last centrifugation step was put in a chloroform-methanol (2:1) extraction mixture. The volume of these control samples was similar to the cercariae sample volume. The sample preparation usually took around 6–7 hours for one sample. The age of the cercariae in our samples was estimated as a period from the start of snail exposition for cercariae shedding to the freezing, lasting ~29 h, except for two samples, which were 51 h and 53 h old (Supporting information). In general, the age was overestimated because some cercariae emerged hours after a snail was placed in fresh water. We could not collect younger cercariae due to the need for high sample concentrations, as recommended in previous studies (McKee et al., 2020). Figure 1. The procedure for collecting D. pseudospathaceum cercariae for lipid analysis. Lipid analysis The total lipids (TL) were extracted using a chloroform-methanol mixture in a 2:1 ratio (v/v) according to the Folch method (Folch et al., 1957). Lipid classes as total phospholipids (PL), monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), cholesterol (Chol) and its esters (Chol esters), waxes, non-esterified fatty acids (NEFA) were identified by high-performance thin-layer chromatography (HPTLC) using HPTLC equipment. Microquantities of the sample (2 μl) were applied by streaking onto glass chromatographic plates HPTLC Silicagel 60 F254 Premium Purity (Merck, USA) using a semiautomatic applicator Linomat 5 (CAMAG, Switzerland). TL was eluted in a sealed chromatographic chamber ADC2 (CAMAG, Switzerland). The mobile phase was a solvent system of hexane:dimethyl ether:acetic acid in a ratio of 32:8:0.8 (v/v) (Olsen & Henderson, 1989). The separation of lipid classes was carried out under the following conditions: the humidity in the system was 47–49% replacing air with eluent vapor in the chromatographic chamber and preconditioning of the chromatographic plate for 10 min. Lipids were stained in a derivatizer (CAMAG, Switzerland) with a solution of copper sulfate (CuSO₄) acidified with orthophosphoric acid (H₃PO₄). Then, the plate was heated to 160 °C for 15 min. Qualitative and quantitative determination of lipid components was performed using a TLC Scanner 4 scanning densitometer (CAMAG, Switzerland) in adsorption mode at a wavelength of 360 nm (Hellwig, 2005). Separate lipid reference standards (Sigma-Aldrich, USA) were applied to identify analyzed lipid classes; the correspondence of mobility coefficients (Rf) was also considered. Qualitative and quantitative analyses of fatty acids (FA) of the TL were conducted using gas chromatography (GC) coupled with either a mass-selective (GC-MSD) or a flame ionization (GC-FID) detector. The qualitative FA composition was determined using a GC-MSD, Maestro-αMS (Saitegra, Russia). Fractionation was carried out for 32 min under gradient thermostatting with the following temperature program: initial temperature of 140 °C (held for 5 min), increasing from 140 °C to 240 °C at a rate of 4 °C/min followed by a final temperature of 240 °C (held for 2 min). A capillary column HP-88 (60 m × 0.25 mm × 0.20 μm) with helium as a mobile phase was used for FA separation. Detection of FA was performed in SIM/SCAN modes: SIM mode for registration of FA included in analytical standards (Supelco 37, Bacterial Acid Methyl Ester (BAME) Mix and PUFA №1 Marine source), and SCAN mode for searching and identification of unique FA components in the range of 50–400 m/z. Each sample was analyzed in two replicates: in Split mode (with a flow division of 1:40) and in Splitless mode (without flow division, used for the determination of trace amounts of substances). The obtained data were analyzed using the Maestro Analyst v. 1.025 software and the NIST library. Quantitative analysis of FA was performed on a GC-FID, Chromatec-Crystal-5000.2 (Chromatec, Russia) under the same chromatographic separation conditions as those used for GC-MSD (with the exception of the mobile phase which was nitrogen instead of helium). The results were processed using the Chromatec-Analytics v. 3.0.298.1 software. The quantitative content of individual FA in the sample was determined by the calibration method. The reference Supelco 37 mixture (Sigma-Aldrich, USA) was used as an external standard. The correction calibration factor (R i ) for the quantitative calculation of FA not included in the Supelco 37 mixture was calculated using the formula (GOST ISO/TS 17764-2-2015, 2017): \begin{equation} R_{i}=\ \frac{M_{r}\left(n_{i}-1\right)}{M_{i}\left(n_{r}-1\right)}\nonumber \\ \end{equation} where M r is the molar mass of the internal standard FA (16:0), g/mol; n i is the number of carbon atoms of the i FA; M i is the molar mass of the i FA, g/mol; n r is the number of carbon atoms of the internal standard FA (16:0). Estimation of dry weight of cercariae Cercariae of D. pseudospathaceum were collected from a mixture derived from 5 snails individually placed in commercial bottled water for 24 h. A known number of cercariae (70–119, mean ± SD = 95 ± 12) were pipetted from the mixture and put in tin cylindrical capsules, 5 mm in diameter and 9 mm in height (NC Technologies, Italy). The empty capsules were weighed before the experiment. As controls, we used water from the cercariae mixture, but without the cercariae, each time they were collected. Collection took around 6 h, hence the maximum age of cercariae was similar to those in lipid samples (~30 h). Then, samples were dried at 60 °C for 66 h and immediately weighted using microbalance (Mettler Toledo XP6, USA). Before weighing, the samples were kept in a sealed container with silica gel to control for humidity. The dry weight of each sample was measured as the difference between the weight of the capsule with the dried sample and the weight of this empty capsule. For each sampling day, we calculate the mean dry weight of controls. To calculate the dry weight of individual cercariae, we subtracted the average dry weight of the control samples from the dry weight of the cercariae samples collected on the same day and divided the result by the number of cercariae in each sample. In total, 31 cercariae samples and 19 control samples were collected over three sampling sessions during three weeks (Supporting information). Data analysis For the analysis of fatty acids composition, we grouped them by length and number of double bonds. Thus, isomers and fatty acids differing by the position of double bonds were assigned to the same group. Additionally, fatty acids with a mean contribution of less than 1% in both temperature treatments were excluded from the analysis (Supporting information). The differences in the amount and composition of lipid classes and fatty acids between cooled and uncooled samples were tested by analysis of similarities (ANOSIM). To visualize the results, we used the non-metric multi-dimensional scaling (nMDS) based on a Bray–Curtis dissimilarity matrices for the absolute values and relative abundances of different lipid groups in samples. The analysis was performed on both untransformed data and data with square root transformation. We performed a one-way ANOVA to test the hypotheses about temperature treatment effects on total lipids and polyunsaturated fatty acids. The Bonferroni–Holm correction was applied to account for the multiple comparisons. A linear model with the Gaussian error structure and identity link function was used to estimate the influence of sampling day and treatment (cercariae vs. control) on the dry sample weight. However, though model residuals distribution did not violate the normality assumption (Shapiro–Wilk test, W = 0.97, p = 0.20), we could not manage the heteroscedasticity problem (Levene’s test, F(5, 44) = 8.0, p = 0.00002). Data transformation did not help. Though it is often suggested that this assumption violation does not influence inference too much, we used robust standard errors obtained with a ‘vcovHC’ function from the ‘sandwich’ package (Colonescu, 2016; Zeileis et al., 2020) from R software (R Core Team, 2023). The program PRIMER 7 (PRIMER-E Ltd, Plymouth) was also used for the data analysis. Results Lipids The estimated number of D. pseudospathaceum cercariae per sample varied between 1845 and 57955 individuals (Supporting information). Even 1845 cercariae (dry weight 269 μg) were sufficient to carry out HPTLC and GC. The ANOSIM did not show a significant difference between cooled and uncooled samples in the composition and amount of lipid classes and fatty acids (table 1). The results of nMDS are available in Supporting information. Cooled and uncooled samples also did not differ by mean proportions of total lipids (one-way ANOVA: F(1, 16) = 1.49, p = 0.24) and PUFA (F(1, 16) = 0.22, p = 0.65). In addition, we did not reveal differences in the absolute values of total lipids (F(1, 16) = 0.005, p = 0.98) and PUFA (F(1, 16) = 1.18, p = 0.29). Therefore, below we provide mean results for the full dataset, including both cooled and uncooled samples (n = 18). Table 1. The results of the analysis of similarity (ANOSIM) for cooled vs. uncooled samples by lipid classes and fatty acids. On average ( ± SD), cercariae of D. pseudospathaceum contained high proportions (% of total lipids) of cholesterol esters, waxes, phospholipids, cholesterol, and TAG (figure 2 and table 2, Supporting information). The most prevalent fatty acids were saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) (37 ± 15% and 37 ± 18%, respectively) (figure 3). Among them, palmitic acid, oleic acid, stearic acid, and 18:1n-5 were common (table 2). Polyunsaturated fatty acids (PUFA) constituted 26 ± 15% of the total, where n-6 PUFA and n-3 PUFA shares were 16 ± 9% and 10 ± 12%, respectively. Among essential fatty acids (EFA) were linoleic acid (LNA), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) (figure 4 and table 2). Figure 2. The proportion of lipid classes in cercariae of D. pseudospathaceum. The height of the boxes represents the interquartile range (IQR), the horizontal line is the median, the whiskers are the highest and lowest values within the 1.5 × IQR, cross (×) is the mean. Phospholipids (PL), monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), cholesterol (Chol) and its esters (Chol esters), free fatty acids (FFA). Table 2. The lipid and fatty acid composition of D. pseudospathaceum cercariae. Only the most common (with a mean share > 3% of the total) and essential fatty acids are listed here; the information about all fatty acids is available in Supporting information. Phospholipids (PL), monoacylglycerols (MAG), diacylglycerols (DAG), triacylglycerols (TAG), cholesterol (Chol) and its esters (Chol esters), free fatty acids (FFA), alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LNA). Figure 3. The proportion of fatty acids in cercariae of D. pseudospathaceum , grouped according to their saturation. The sum of saturated fatty acids (ΣSFA), monounsaturated fatty acids (ΣMUFA), n-3 polyunsaturated fatty acids (Σn-3 PUFA), n-6 polyunsaturated fatty acids (Σn-6 PUFA). For the plot details, see Figure 2. Figure 4. The proportion of essential fatty acids in cercariae of D. pseudospathaceum . Alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LNA). For the plot details, see Figure 2. An outlier from the LNA sample (34%) was excluded from the plot to improve data visualisation. The amount of fatty acids in cercariae samples was compared with those in control samples (supernatant collected at the last centrifugation step) to assess whether they could be lost during centrifugation. The concentration of fatty acids (μg/ml) was significantly lower in the controls than in corresponding cercariae samples (paired t-test, t > 2.2; df = 38, p < 0.05 in all four cases) (Supporting information). In rare occasions (8%, 13 of 156 cases), some fatty acids (e.g., saturated heneicosanoic, arachidic, and lignoceric acids) were more abundant (up to 7 times) in control than in cercariae samples. Excluding these rare peaks, the concentrations of certain fatty acids in controls averaged 6–20% of those in the cercariae samples. Therefore, we might expect that underestimation of fatty acid concentrations in the D. pseudospathaceum cercariae due to loss during centrifugation does not exceed 20% (Supporting information). Dry weight Control samples were significantly lighter than those with cercariae (estimate ± SE = −10.4 ± 1.67 μg, t = −6.2, p < 0.0001, Supporting information). On average, the dry weight of control samples constituted 84–90% of the dry weight of cercariae samples on different sampling days. Therefore, the contribution of organic matter released by snails and other contaminants in water with cercariae was high (Supporting information). Sample weights differed between days. The mean (± SD) dry weight of individual cercariae was 0.146 ± 0.084 μg (in total 2370 cercariae were weighted, excluding the 6 samples that gave negative weight values after correction for control means). For detailed information on dry weight estimation and the linear regression plot see Supporting information. Discussion To our knowledge, this is the first study to investigate the effects of short-term cooling on lipids in live aquatic animals. Utilization of energy stores (i.e., glycogen) and cercarial swimming speed generally decrease with temperature (Lyholt & Buchmann, 1996; Morley, 2011, 2020), suggesting overall lower metabolic activity. Therefore, we hypothesized that lipids would be utilized and degraded slower at lower temperatures, resulting in higher levels of total lipids in cooled samples. However, we found that cooling for ~4 hours hours (up to 0 °C) had no significant effect on the composition and amount of each lipid class of D. pseudospathaceum cercariae. Contrary to our hypothesis, low temperatures, which often increase fatty acid unsaturation in animals (Hazel & Williams, 1990; Hazel, 1995; Guschina & Harwood, 2006), did not have this effect on cercariae. Since cooling had no effect on lipidome, cercariae likely do not adjust their lipid composition to low temperatures or may do so differently from other animals. This may be explained by the fact that cercariae rarely meet low temperatures in nature because in temperate latitudes they are typically released by snails at > 10 °C (Morley, 2012) and can occasionally occur in cold deep waters (e.g., settling because of cercariae aging or movement of the snail host). Additionally, the duration of cooling was probably insufficient to induce changes. However, considering the short life span (Morley, 2012) of cercariae and the high turnover rates of lipids in parasites (Brouwers et al., 1997) several hours of cooling appear to be enough to evaluate the effect of temperature on lipids of these small and active organisms. Our findings suggest that short-term cooling can be an effective method for concentration of D. pseudospathaceum cercariae for lipid analyses, as it does not significantly alter their lipid composition. The lipidome of the widespread trematodes of the family Diplostomidae was previously unknown. Our study showed that D. pseudospathaceum cercariae are rich in neutral lipids and palmitic, oleic and stearic acids as other parasitic and free-living invertebrates (Arts, 1999; Marsit et al., 2000, 2000a; Muller et al., 1999, Schariter et al., 2002; Fokina et al., 2018; Giera et al., 2018; McKee et al., 2020). Essential fatty acids like LNA, ALA, EPA, and DHA, which indicate nutritional quality and ecosystem productivity, are also abundant in D. pseudospathaceum as in other cercariae (Giera et al., 2018; Fokina et al., 2018; McKee et al., 2020). Unique fatty acids (e.g., 18:1n-5) were identified in D. pseudospathaceum cercariae, while others, like arachidonic acid (ARA), were absent despite being detected in earlier cercariae studies (Giera et al., 2018; Fokina et al., 2018; McKee et al., 2020). Cercariae of D. pseudospathaceum were also distinguished from other trematode species by relatively high levels of triacylglycerols (Marsit et al., 2000, 2000a; Schariter et al., 2002; Fokina et al., 2018) and waxes, a lipid class previously unreported for trematodes. These lipids may regulate buoyancy (Campbell & Dower, 2003; Lee et al., 2006), be crucial for cercariae survival or their transformation into the next stage. The energetic role of waxes and TAG in cercariae is questionable (Fried et al., 1998; Bexkens et al., 2019). Differences in lipidomes across species of cercariae can be attributed to various factors, such as specificity of cercariae morphology, behavior, life-history strategy, the environmental background of their hosts or methodological features (Beers et al., 1995; Ferreira et al., 2014; McKee et al., 2020; Babaran et al., 2021; Wangchuk et al., 2023). Further we emphasize some important issues in cercariae sampling and provide methodological considerations. The number of cercariae used for one sample differed by orders of magnitude in different species and studies, ranging from 17 to 60000 individuals (Smith et al., 1966; Marsit et al., 2000; Schariter et al., 2002; McKee et al., 2020). The smaller the cercariae, the more of them are needed. In our study, 1845 cercariae of ~390 µm in length (Faltýnková, 2007; Selbach et al., 2015; Mironova et al., 2020) were enough for HPTLC and GC analysis. In terms of dry weight, 0.269 mg of cercariae, significantly less than the previously suggested 15 mg (McKee et al., 2020), is sufficient. This finding is crucial, as it reduces the collection effort, enables larger sample sizes, and allows the collection of sufficient material from a single snail. It can facilitate the investigation of intraspecific variation in the lipidome of parasites. According to our results, when collecting cercariae by centrifugation, about 6–20% of fatty acids observed in cercariae sample could be removed with supernatant. However, this loss may be overestimated because supernatant may contain lipids originating not only from damaged cercariae but also from microorganisms and snail hosts. Perhaps, mesh filtration can be a more delicate and useful method for the concentration of larger cercariae species, while for D. pseudospathaceum and similar-sized cercariae, centrifugation seems to be the optimal approach. Our study is among the first to provide information about the amounts of lipids and fatty acids per unit of cercariae dry weight. Reporting lipid values in dry weight of cercariae or presenting information about the dry weight of studied objects improves the comparability of lipid data. However, only one earlier study (McKee et al., 2020) provides data on fatty acids composition in such a convenient form, while others used the shares of total lipids, the amount of lipids per one cercaria (Smith et al., 1966; Muller et al., 1999; Marsit et al., 2000, 2000a; Schariter et al., 2002) or other measurement units (e.g., Maloney et al., 1990; Giera et al., 2018). To our knowledge, there are still only single reports on the dry weight of cercariae besides the present study (Lambden & Johnson, 2013; Yurlova, 2016). Additionally, host and parasite variability can affect cercarial lipid content. Therefore, information about the number of hosts and their environmental background is necessary for the correct interpretation of lipid data. Thus, collecting cercariae from multiple hosts ensures average estimates, but only one study explicitly provides data on the number of mollusc hosts (McKee et al., 2020). The age of cercariae can also contribute to a lipid composition. The biochemical composition of cercariae is likely to change over time and, hence, influence their survival and infectivity (Karvonen et al., 2003; Whitfield et al., 2003). Few studies specify the age of cercariae, which ranges from ~3 hours (Maloney et al., 1990) to over 8 hours (Marsit et al., 2000, 2000a; Schariter et al., 2002; McKee et al., 2020). In our study, the maximum age of D. pseudospathaceum cercariae was ~29 hours in most of the samples. Thus, the amount of lipids may be underestimated in our study, as cercariae could utilize some of them. The variation in age is particularly relevant when considering storage lipids that serve as energy sources in many organisms (Gurr et al., 2002) and can be utilized over time. However, the effect of aging on cercariae lipids has not been studied explicitly yet. In conclusion, this study provides new insights into the lipid composition of cercariae and the effects of short-term cooling on it. Cooling did not significantly alter the lipid and fatty acid profiles of cercariae and, therefore, can be recommended for cercariae concentration during sampling. The lipidome of D. pseudospathaceum cercariae shares some similarities with the lipidomes of other invertebrates. Notably, it contains unique lipid classes, such as waxes, which have not been previously reported in trematode cercariae. These findings advance understanding of cercarial lipid biochemistry and offer methodological improvements in cercariae sampling. These findings advance our understanding of cercarial lipid biochemistry and offer methodological improvements in cercariae sampling, which will be useful for future studies on the influence of aging, host, and environment on parasite lipids. References 1. Lee HW, Ban S, Ikeda T, Matsuishi T. 2003 Effect of temperature on development, growth and reproduction in the marine copepod Pseudocalanus newmani at satiating food condition. J. Plankton. Res. 25:261–271. ( Lee SH, Lee MC, Puthumana J, Park JC, Kang S, Han J, Shin K, Park HG, Om A, Lee J. 2017 Effects of temperature on growth and fatty acid synthesis in the cyclopoid copepod Paracyclopina nana . Fish. Sci. 83:725–734. ( Guschina IA, Harwood JL. 2006 Mechanisms of temperature adaptation in poikilotherms. FEBS Lett. 580, 5477–5483. ( Morley NJ. 2011 Thermodynamics of cercarial survival and metabolism in a changing climate. Parasitology 138, 1442–1452. ( Wu G, Baumeister R, Heimbucher T. 2023 Molecular mechanisms of lipid-based metabolic adaptation strategies in response to cold. Cells 12, 1353. ( Hazel JR, Williams EE. 1990 The role of alterations in membrane lipid composition in enabling physiological adaptation of organisms to their physical environment. Prog. Lipid Res. 29:167–227. ( Hazel JR. 1995 Thermal adaptation in biological membranes: is homeoviscous adaptation the explanation? Annu. Rev. Physiol. 57:19–42. ( Jones AL, Hann AC, Harwood JL, Lloyd D. 1993 Temperature-induced membrane-lipid adaptation in Acanthamoeba castellanii . Biochem. J. 290:273–278. ( Murata N. 1989 Low-temperature effects on cyanobacterial membranes. J. Bioenerg. Biomembr. 21, 61–75. ( Nanton DA, Castell JD. 1999 The effects of temperature and dietary fatty acids on the fatty acid composition of harpacticoid copepods, for use as a live food for marine fish larvae. Aquaculture 175, 167–181. ( Schlechtriem C, Arts MT, Zellmer ID. 2006 Effect of temperature on the fatty acid composition and temporal trajectories of fatty acids in fasting Daphnia pulex (Crustacea, Cladocera). Lipids 41, 397–400. ( Pond DM, Tarling GA, Mayor DJ. 2014 Hydrostatic pressure and temperature effects on the membranes of a seasonally migrating marine copepod. PLoS One 9, e111043. ( Lee MC, Yoon DS, Lee Y, Choi H, Shin KH, Park HG, Lee JS. 2020 Effects of low temperature on longevity and lipid metabolism in the marine rotifer Brachionus koreanus . Comp. Biochem. Physiol. A. Mol. Integr. Physiol. 250, 110803. ( Morris RJ. 1972 The preservation of some oceanic animals for lipid analysis. J. Fish. Res. Bd. Canada 29, 1303–1307. Ohman MD. 1996 Freezing and storage of copepod samples for the analysis of lipids. Mar. Ecol. Prog. Ser. 130, 295–298. ( Schariter JA, Pachuski J, Fried B, Sherma J. 2002 Determination of neutral lipids and phospholipids in the cercariae of Schistosoma mansoni by high performance thin layer chromatography. J. Liq. Chromatogr. Relat. Technol. 25, 1615–1622. ( Lawson JR, Wilson RA. 1980 The survival of the cercariae of Schistosoma mansoni in relation to water temperature and glycogen utilization. Parasitology 81, 337–348. ( Pechenik JA, Fried B. 1995 Effect of temperature on survival and infectivity of Echinostoma trivolvis cercariae: a test of the energy limitation hypothesis. Parasitology 111, 373–378. Frayha GJ, Smyth JD. 1983 Lipid metabolism in parasitic helminths. Adv. Parasitol. 22, 309–387. ( Fokina N, Ruokolainen T, Bakhmet I. 2018 Lipid profiles in Himasthla elongata and their intermediate hosts, Littorina littorea and Mytilus edulis . Mol. Biochem. Parasitol. 225, 4–6. ( Fried B, Eyster LS, Pechenik JA. 1998 Histochemical glycogen and neutral lipid in Echinostoma trivolvis cercariae and effects of exogenous glucose on cercarial longevity. J. Helminthol. 72, 83–85. ( Bexkens ML, Mebius MM, Houweling M, Brouwers JF, Tielens AGM, van Hellemond JJ. 2019 Schistosoma mansoni does not and cannot oxidise fatty acids, but these are used for biosynthetic purposes instead. Int. J. Parasitol. 49, 647–656. ( Furlong ST, Caulfield JP. 1988 Schistosoma mansoni : sterol and phospholipid composition of cercariae, schistosomula, and adults. Exp. Parasitol. 65, 222–231. ( Marsit CJ, Fried B, Sherma J. 2000 Neutral lipids in cercariae, encysted metacercariae, and rediae of Echinostoma caproni . J. Helminthol. 74, 365–367. ( Smith TS, Brooks TJ, White HB. 1966 Thin-layer and gas-liquid chromatographic analysis of lipid from cercariae of Schistosoma mansoni . Am. J. Trop. Med. Hyg. 15, 307–313. ( Muller EE, Fried B, Sherma J. 1999 HPTLC determination of neutral lipids in the cercariae of Echinostoma trivolvis and Echinoparyphium sp. (Trematoda). J. Planar. Chromatogr. -Mod. TLC 12, 306–308. Marsit CJ, Fried B, Sherma J. 2000a Neutral lipids in cercariae, encysted metacercariae, and rediae of Zygocotyle lunata . J. Parasitol. 86, 1162–1163. ( Giera M, Kaisar MMM, Derks RJE, Steenvoorden E, Kruize YCM, Hokke CH, Yazdanbakhsh M, Everts B. 2018 The Schistosoma mansoni lipidome: leads for immunomodulation. Anal. chim. acta 1037, 107–118. ( McKee KM, Koprivnikar J, Johnson PTJ, Arts MT. 2020 Parasite infectious stages provide essential fatty acids and lipid-rich resources to freshwater consumers. Oecologia 192, 477–488. ( Johnson PT, Dobson A, Lafferty KD, Marcogliese DJ, Memmott J, Orlofske SA, Poulin R, Thieltges DW. 2010 When parasites become prey: ecological and epidemiological significance of eating parasites. Trends Ecol. Evol. 25, 362–71. ( Mironova E, Gopko M, Pasternak A, Mikheev V, Taskinen J. 2019 Trematode cercariae as prey for zooplankton: effect on fitness traits of predators. Parasitology 146, 105–111. ( Mironova E, Gopko M, Pasternak A, Mikheev VN, Taskinen J. 2020 Cyclopoids feed selectively on free‐living stages of parasites. Freshw. Biol. 65, 1450–1459. ( Lafferty KD et al. 2008 Parasites in food webs: the ultimate missing links. Ecol. Lett. 11, 533–546. ( Maloney MD, Semprevivo LH, Coles GC. 1990 A comparison of the glycolipid compositions of cercarial and adult Schistosoma mansoni and their associated hosts. Int. J. Parasitol. 20, 1091–1093. ( Shigin A. 1986 Trematody Fauny SSSR. Rod Diplostomum. Metacercarii . Moscow: Russia. Nauka. (in Russian) Valtonen ET, Gibson DI. 1997 Aspects of the biology of diplostomid metacercarial (Digenea) populations occurring in fishes in different localities of Northern Finland. Ann. Zool. Fenn. 34, 47–59. Faltýnková A, Nasincová V, Kablásková L. 2007 Larval trematodes (Digenea) of the great pond snail, Lymnaea stagnalis (L.), (Gastropoda, Pulmonata) in Central Europe: a survey of species and key to their identification. Parasite 14, 39–51. ( Folch J, Lees М, Sloane Stanley GH. 1957 A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226, 497–509. Olsen RE, Henderson RJ. 1989 The rapid analysis of neutral and polar marine lipids using double-development HPTLC and scanning densitometry. J. Exp. Mar. Biol. Ecol. 129, 189–197. ( Hellwig J. 2005 Defining parameters for a reproducible TLC-separation of phospholipids using ADC 2. Ph.D. thesis, University of Applied Sciences Northwestern Switzerland (FHNW), Windisch, Switzerland. GOST ISO/TS 17764-2-2015. 2017 Feed, compound feed. Determination of fatty acid content. Part 2. Gas chromatographic method. Colonescu C. 2016 Principles of econometrics with R. (https://bookdown.org/ccolonescu/RPoE4/). Zeileis A, Köll S, Graham N. 2020 Various versatile variances: an object-oriented implementation of clustered covariances in R. J. Stat. Softw. 95, 1–36. ( R Core Team. 2013 R: a language and environment for statistical computing. Vienna, Austria: R Foundation for Statistical Computing. (https://www.R-project.org). Clarke KR, Gorley RN. 2015 PRIMER v7: User Manual/Tutorial. PRIMER-EPlymouth. (www.primer-e.com/software). Lyholt HC, Buchmann K. 1996 Diplostomum spathaceum : effects of temperature and light on cercarial shedding and infection of rainbow trout. Dis. Aquat. Organ. 25, 169–173. ( Morley NJ. 2020 Cercarial swimming performance and its potential role as a key variable of trematode transmission. Parasitology 147, 1369–1374. ( Morley NJ. 2012 Cercariae (Platyhelminthes: Trematoda) as neglected components of zooplankton communities in freshwater habitats. Hydrobiologia 691, 7–19. ( Brouwers JF, Smeenk IM, van Golde LM, Tielens AG. 1997 The incorporation, modification and turnover of fatty acids in adult Schistosoma mansoni . Mol. Biochem. Parasitol. 88, 175–185. ( Arts MT. 1999 Lipids in freshwater zooplankton: selected ecological and physiological aspects . In Lipids in freshwater ecosystems (eds MT Arts, BC Wainman), pp. 71–90. New York, NY: Springer. ( Campbell RW, Dower JF. 2003 Role of lipids in the maintenance of neutral buoyancy by zooplankton. Mar. Ecol. Prog. Ser. 263, 93–99. ( Lee RF, Hagen W, Kattner G. 2006 Lipid storage in marine zooplankton. Mar. Ecol. Prog. Ser. 307, 273–306. ( Beers KW, Fried B, Fujino T, Sherma J. 1995 Effects of diet on the lipid composition of the digestive gland-gonad complex of Biomphalaria glabrata (Gastropoda) infected with larval Echinostoma caproni (Trematoda). Comp. Biochem. Physio.l B Biochem. Mol. Biol. 110, 729–37. ( Ferreira MS, de Oliveira DN, de Oliveira RN, Allegretti SM, Catharino RR. 2014 Screening the life cycle of Schistosoma mansoni using high-resolution mass spectrometry. Anal. Chim. Acta 845, 62–9. ( Babaran D, Koprivnikar J, Parzanini C, Arts MT. 2021 Parasites and their freshwater snail hosts maintain their nutritional value for essential fatty acids despite altered algal diets. Oecologia 196, 553–564. ( Wangchuk P, Yeshi K, Loukas A. 2023 Metabolomics and lipidomics studies of parasitic helminths: molecular diversity and identification levels achieved by using different characterisation tools. Metabolomics 19, 63. ( Selbach C, Soldánová M, Georgieva S, Kostadinova A, Sures B. 2015 Integrative taxonomic approach to the cryptic diversity of Diplostomum spp. in lymnaeid snails from Europe with a focus on the ‘ Diplostomum mergi ’ species complex. Parasit. Vectors 8, 300. ( Lambden J, Johnson PT. 2013 Quantifying the biomass of parasites to understand their role in aquatic communities. Ecol. Evol. 3, 2310–2321. ( Yurlova NI. 2016 Productivity and biomass of trematode (Digenea) parasites in lake ecosystems. Dokl. Biol. Sci. 466, 28–31. ( Karvonen A, Paukku S, Valtonen ET, Hudson PJ. 2003 Transmission, infectivity and survival of Diplostomum spathaceum cercariae. Parasitology 127, 217–24. ( Whitfield PJ, Bartlett A, Khammo N, Clothier RH. 2003 Age-dependent survival and infectivity of Schistosoma mansoni cercariae. Parasitology 127, 29–35. ( Gurr MI, Harwood JL, Frayn KN. 2002 Lipid Biochemistry. An Introduction , 5th ed. Oxford: Blackwell Science. Crossref Google Scholar Information & Authors Information Version history V1 Version 1 14 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cercariae dry weight essential fatty acids lipidome trematode Authors Affiliations Kseniia Savina 0000-0002-9246-5475 [email protected] A N Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences View all articles by this author Misha Gopko 0000-0002-1525-6557 A N Severtsov Institute of Ecology and Evolution of the Russian Academy of Sciences View all articles by this author Viktor Voronin FSBIS Karelian Research Centre Russian Academy of Sciences View all articles by this author Svetlana Murzina FSBIS Karelian Research Centre Russian Academy of Sciences View all articles by this author Ekaterina Mironova 0000-0002-9557-6031 A N Severtsov Institute of Ecology and Evolution RAS View all articles by this author Metrics & Citations Metrics Article Usage 176 views 110 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Kseniia Savina, Misha Gopko, Viktor Voronin, et al. 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